Chlamydomonas reinhardtii is being transformed from a model organism to an industrial organism for the production of pigments, fatty acids, and pharmaceuticals. Genetic modification has been used to increase the economic value of C. reinhardtii. However, low gene-editing efficiency and position-effects hinder the genetic improvement of this microorganism. Recently, site-specific double-stranded DNA cleavage using CRISPR-Cas9 system has been applied to regulate a metabolic pathway in C. reinhardtii. In this study, we proved that site-specific gene expression can be induced by CRISPR-Cas9-mediated double-strand cleavage and non-homologous end joining (NHEJ) mechanism. The CRISPR-Cas9-mediated knock-in method was adopted to improve gene-editing efficiency and express the reporter gene on the intended site. Knock-in was performed using a combination of ribonucleoprotein (RNP) complex and DNA fragment (antibiotics resistance gene). Gene-editing efficiency was improved via optimization of a component of RNP complex. We found that when the gene CrFTSY was targeted, the efficiency of obtaining the desired mutant by the knock-in method combined with antibiotic resistance was nearly 37%; 2.5 times higher than the previous reports. Additionally, insertion of a long DNA fragment (3.2 and 6.4 kb) and site-specific gene expression were analyzed. We demonstrated the knock-out phenotype of CrFTSY and on-site inserted gene expression of luciferase and mVenus at the same time. This result showed that CRISPR-Cas9-mediated knock-in can be used to express the gene of interest avoiding position-effects in C. reinhardtii. This report could provide a new perspective to the use of gene-editing. Furthermore, the technical improvements in genetic modification may accelerate the commercialization of C. reinhardtii. Chlamydomonas reinhardtii is widely used as a model organism and considered to be a potential cell factory to produce value-added compounds  containing a 20-base pair sequence complementary to the target DNA, and a trans-activating crRNA (tracrRNA). crRNA and tracrRNA can be physically linked to form single guide RNA (gRNA)  system has been developed  and transcription activator-like effector nuclease (TALEN) are used to create specific double-stranded DNA cleavages . HoweverA (gRNA) . In C. reveloped . CRISPR-eveloped , and is eveloped . Countereveloped . Howevereveloped .C. reinhardtii. However, the efficiency of these methods is low and the rate of unintended mutations is high. In this study, to develop the new technique of genetic modification in C. reinhardtii, we investigated the knock-in method to improve gene-editing efficiency while inducing gene expression at the desired location. Phenotypic studies indicated that the two desired characteristics were obtained simultaneously. We optimized the technique that increased gene-editing efficiency to >30% and successfully demonstrated site-specific gene expression by avoiding random mutations.As mentioned above, advanced gene expression tools and gene-editing techniques have played a major role in increasing the commercial use of Chlamydomonas reinhardtii CC4349, CC124, and CC503  were maintained photoheterotrophically in Tris-Acetate-Phosphate (TAP) medium at 25°C with continuous light (80 μmol photons m–2 s–1) on an orbital shaker (100 rpm). The cells were cultivated till the log phase in liquid TAP medium under the same conditions for all the experiments. For the selection and maintenance of transformant lines, solid TAP medium fortified with 30 μg/mL hygromycin-B  for ΔCrFTSY-Ga and with 30 μg/mL hygromycin-B, 30 μg/mL paromomycin  for ΔCrFTSY-mV, were used.CrFTSY and the gRNA sequence, 5′-CGATCTTCAGAGCAGTGCGG-3′, that was the same as that of the previous study  following the manufacturer’s protocol. The inserted DNA fragments, aph7 gene  (1.6 kb) (aph7-GLuc (Gaussia Luciferase) (3.2 kb)  – mVenus (yellow fluorescence protein) – aph7 (6.4 kb) . All the inserted DNA fragments were purified by agarose gel extraction.To optimize the gene-specific knock-out efficiency, RNP method was used and the optimal conditions were determined with slight modification according to the method described previously . The tarus study , were us(1.6 kb)  left and(3.2 kb)  DNA cass(6.4 kb)  DNA cassChlamydomonas, 2 × 107 cells were transformed with Cas9 protein premixed with gRNA (RNP complex). Lyophilized Cas9 protein was dissolved in gRNA solution prepared with nuclease-free water and the mixture was incubated for 10 min at room temperature (20–25°C) to make a complex. Cells mixed with the RNP complex were transferred into 4 mm cuvettes and transformed by Gene Pulser XcellTM Electroporation Systems  set at 600 V, 50 μF, and 200 Ω. After transformation, the cells were incubated overnight (16–20 h) and spread on the selective medium. Colonies appeared within 2 weeks. The antibiotic resistance colonies were transferred to liquid medium for the sequence analysis. This pre-selected colony number is classified as “candidate” for the calculation of knock-in efficiency.To generate the target-specific knock-in mutant using RNP in The genomic DNA was prepared from the colonies grown on selective medium using the Chelex-100 method . Targete6 cells were deposited on solid TAP medium. The cells were incubated for 24 h and the difference in color characteristics was compared. For measurement of the chlorophyll contents, 1 × 107 cells were suspended in 80% (v/v) acetone, and the absorbance was measured by spectrophotometer at wavelength of 663 and 647 nm. The formula used for calculating the content is given by Wild-type and mutant cells were cultured in liquid TAP medium untill the mid-log phase. To make comparison of colorimetric analysis, the cultured cells were collected from the liquid media, and 2.5 × 10GLuc expression. GLuc is secreted in the medium hence, its activity was measured using the complete cell culture . The cells were suspended in 40 μL of lysis buffer and lysed by vigorous vortexing. The lysed cells were pelleted by centrifugation at 13,000 rpm for 5 min. Then, 80 μL supernatant was mixed with equal volume of substrate solution. The chemiluminescence was measured immediately after mixing the two solutions in a Glo MaxTM 20/20 luminometer . The statistical difference was analyzed by Student’s t-test .Luciferase activity was measured by a previously reported protocol  with sli culture  and it iΔCrFTSY-mV was cultivated in selective medium as mentioned above. Five microliters of cultured cells were dropped on a glass slide and covered with a coverslip. Fluorescence was detected by a fluorescence microscope . Fluorescence detection wavelengths were 540 ± 20 nm with YFP filter for mVenus and 630 ± 30 nm with Texas RED filter for auto-fluorescence of chlorophyll. The magnification was 400×.PvuII or SmaI restriction enzymes. Digested genomic DNA was separated using 0.8% agarose gel and transferred to a nylon membrane by capillary transfer. Membrane attached DNA was developed through hybridization with an aph7 probe and amplified by PCR with specific primers  using the Gene Images AlkPhos Direct Labeling and Detection System . The process was carried out according to the manufacturer’s protocol.Southern blot was used to confirm single DNA insertion in mutants. Genomic DNA was extracted from cells in the log phase by the CTAB method . The gen6) were boiled in SDS-PAGE loading buffer, electrophoresed on 15% SDS-polyacrylamide gels, and transferred to a PVDF membrane using Xcell II blot module . GLuc antibody  was used to detect GLuc protein. ATP-β antibody  was used as a reference. HRP-conjugated goat anti-rabbit IgG (H + L) antibody  was used as a secondary antibody. GLuc and ATP-β were visualized on an X-ray film by chemiluminescence using EPD Western Reagent .Cultivated cells  was observed to be maximum at 36.8%, when transformed with 100 μg Cas9 and 70 μg gRNA. Even on transformation with 10 μg Cas9 and 7 μg gRNA, ΔCrFTSY efficiency was >16.5%, which was higher than previously reported  and CC124  were observed under the same method and it was found to be similar to that of C. reinhardtii CC4349 . Therefore, in this study gene-editing efficiency was analyzed by using different concentrations of RNP complex required for transformation . Every sreported . Thus, wi CC4349 . This reC. reinhardtii. This DNA integration in the genome predominantly leads to unexpected mutations and position-effects  and -b (1.34 ± 0.53 nmol mL–1) in ΔCrFTSY-Ga mutants were reduced to 63 and 38% of wild type chlorophyll-a and chlorophyll-b (7.63 ± 0.43 and 3.53 ± 0.83), respectively. Therefore, the chlorophyll a/b ratio was increased by 1.8 ± 0.2-fold in ΔCrFTSY-Ga mutants compared to that of wild type, as also shown in the previous report  found in random integration mutants  of ΔCrFTSY_mV in which obtained . From thobtained . Two mutobtained , In/Del obtained . Hence, C. reinhardtii requires improvement of the strain by genetic modification for the production of high-value compounds. C. reinhardtii has been extensively studied for fundamental research and industrial use based on its genome sequence data and well-developed molecular tool kit  through counter selection without the use of antibiotic genes . Howeveraph7) as donor DNA to ensure high selection efficiency for the optimization of the Cas9-mediated knock-in method. In contrast to the previously reported CRISPR-Cas9-mediated knock-out methods, the use of a selective marker in the knock-in method of our study enhanced the knock-out efficiency by inserting of an external DNA into the cleaved site (AGP and LCYE) (unpublished data). Therefore, we suggest that the use of antibiotic genes as donor DNA is an efficient method when using the RNP-based gene-editing. In addition, through the optimization of RNP complex used in this study, the gene-editing efficiency was increased up to 37%  and the fact that the single copy of DNA insert was integrated into genome showed that our strategy of avoiding position-effect and expressing the gene of interest at the target location successful (C. reinhardtii improvement studies.An additional benefit of establishing the knock-in method is that it avoids the position-effects of random mutations that occur during the transformation process. We strategically utilized this methodology to validate the expression of the gene of interest at the desired position . The succcessful . The sucIn this paper, we reported an improved gene-editing technique. Despite several improvements in different techniques, researchers still face the problem of tedious processes for species improvement. Even though the target-oriented gene insertion based on knock-in method was performed, validation for the precise knock-in was still required. Besides, due to the lack of information on the sites effectively targeted by Cas9, the location of gene insertion is limiting, which unavoidably involves the deletion of one gene. Therefore, it is important to discover target locations with high gene-editing efficiency without affecting the biological function of the cell. As our results show in The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.EJ and JK conceived and designed the experiments. KB confirmed the optimal condition of RNP. JK and SL performed the knock-in study and analyzed the gene expression. JK, SL, and EJ wrote the manuscript. All authors reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Moreover, patients with metastatic disease presented higher levels of EV-derived TIMP-1 mRNA when compared with patients with localized disease (p = 0.002) and when we stratified those patients in high and low levels of TIMP-1 EV-derived mRNA, the ones presenting higher levels had a lower overall survival (p = 0.030). EV-derived TIMP-1 mRNA may be a good prognostic biomarker candidate for ccRCC.The tumor microenvironment has gained a lot of attention from the scientific community since it has a proven impact in the development of tumor progression and metastasis. Extracellular vesicles (EVs) are now considered one of the key players of tumor microenvironment modulation. Clear cell renal cell carcinoma (ccRCC) is the most lethal urological neoplasia and presents a high metastatic potential, which reinforces the need for the development of more effective predictive biomarkers. Our goal was to evaluate the applicability of EV-derived matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) as prognostic biomarkers for ccRCC. To do so, we studied the plasma EV content of 32 patients with localized ccRCC and 29 patients with metastatic ccRCC. We observed that patients with localized disease and tumors larger than 7 cm presented higher levels of plasma EV-derived TIMP-1 mRNA when compared with patients presenting smaller tumors ( Cancer is a heterogeneous disease, with different etiology and natural history, that develops through interactions between environmental and genetic factors, involving the deregulation of multiple pathways responsible for the fundamental cell processes, such as proliferation, differentiation, migration and cell death . HoweverMMPs are a family of 23 zinc-dependent endopeptidases that are crucial for ECM degradation and the processing of cell surface molecules . On the Renal cell carcinoma (RCC) is the most common solid cancer of the adult kidney and also the most lethal urological cancer . The mos+ vesicular structures  indicated that the vast majority of isolated EVs presented a size range between 50 and 200 nm, which is consistent with the size of exosomes and small microvesicles A–E. The ructures .In order to evaluate the MMPs and TIMPs protein content in EVs derived from HKC-8, 786-O, Caki-1 and RCC-FG2 cell lines we did a multiplexed MMP/TIMP array. The MMP/TIMP array map is displayed in p = 0.089)  B. As for= 0.089) C. TIMP-2= 0.089) D.The 786-O cell line presented the highest MMP-1 intracellular mRNA levels when compared to the other cell lines, which is in agreement with the results of the MMP/TIMP array E HoweverMMP-1 in the cell lines and cell-derived EVs, we proceeded to the quantification of the same mRNAs in the EVs derived from plasma samples of ccRCC patients. We observed that patients with metastatic disease presented higher TIMP-1 EV mRNA levels compared to patients with localized disease (p = 0.002) (p = 0.020) (p = 0.030)  A. When w= 0.020) B. Next, = 0.030) C.p = 0.338) nor in terms of tumor size in the patients presenting localized disease (p = 0.477) (p = 0.013)  D,E. When= 0.013) F. Howevep = 0.028) and ex-smokers (p = 0.053). We did not observe any statistical differences between EV-derived TIMP-1 mRNA and hypertension (p = 0.957) or diabetes mellitus (p = 0.461). The low number of samples where EV-derived TIMP-2 mRNA was detected was not sufficient to allow any statistical analysis using these clinical pathological characteristics (data not shown). The same applies to MMP-1 EV-derived mRNA expression that was only detected in four samples in total (1 from Group A + 3 from Group B) and did not allow any statistical analysis (data not shown). The percentages of TIMP-1, TIMP-2 and MMP-1 mRNA detection in patients’ EVs are presented in Regarding the other clinical pathological characteristics, we observed that patients who smoke had higher levels of EV-derived TIMP-1 mRNA when compared to non-smokers , two of them developed metastasis during the follow-up period. Interestingly, both of them presented high levels of EV-derived TIMP-1 mRNA both in the pre- and post-surgery samples , and both of them underwent radical nephrectomy. The patients’ clinical data are presented in Taking this into account, we further investigated the clinical data of the ccRCC metastatic patients that presented high levels of TIMP-1 mRNA and are currently deceased from the disease. From the seven patients that were deceased, three of them presented metastasis at the moment of diagnosis and the remaining four presented localized disease when they were diagnosed but passed away during a five-year period. These four patients presented at least two recurrences each during this period, which is consistent with a more aggressive disease.MMP-2 activation, which is well known for its impact on cancer progression [MMPs are one of the major classes of proteolytic enzymes involved in tumor invasion and metastasis establishment and their action can be modulated by TIMPs. Despite earlier studies having demonstrated that TIMPs had antimetastatic effects, during the last few years, reports started to indicate a dual function of these proteins, with a positive correlation between their expression and a poorer outcome in several human cancers ,24,25,26gression ,31.In order to get more insight into the EV-derived MMP/TIMP profile in ccRCC, we started with the study of EVs derived from four different renal cell lines. All of the cell lines exhibited different EV-derived MMP/TIMPs protein profiles, with the most prominent differences in the expression of MMP-1, TIMP-1 and TIMP-2. These results highlighted the importance of the intratumor heterogeneity in ccRCC, since different ccRCC clones presented different EV-derived MMP/TIMP profiles and may have different impact on the disease evolution. An example of that was what we observed for the two metastatic ccRCC cell lines, Caki-1 and RCC-FG2, were the Caki-1 cells presented higher protein expression of TIMP-1 and TIMP-2 than RCC-FG2.MMP-1 mRNA was detectable in 786-O EVs. It is important to note that mRNA molecules are known to be unstable and prone to degradation and their incorporation into EVs is described as the only mechanism that allows their stable circulation in body fluids without the risk of degradation by RNase activity, making them very attractive molecules to study in circulating EVs [Next, we focused on the mRNA levels of MMP-1, TIMP-1 and TIMP-2 intracellularly and in the shedded EVs from all the cell lines studied, in order to see if there were also differences in the expression patterns. We observed that TIMP-1 and TIMP-2 mRNAs were present in all the cell lines EVs whereas ting EVs . We furtTIMP-1 mRNA, we observed that both patients presented high levels before surgery and those levels remained high one month after surgery. Although they are speculative and require further validation, these results suggest that EV-derived TIMP-1 mRNA may play a role in disease progression, even when the primary tumor is removed. It is important to note that tumor growth and invasion are also supported by non-cancerous stromal cells, which include endothelial cells, fibroblasts, pericytes and immune cells, all of which are also able to secrete EVs [During the course of follow-up, two of the patients with localized disease (group A) developed metastasis several months after surgery. When we looked up to the levels of EV-derived rete EVs ,34,35. Trete EVs ,36,37. Orete EVs . It has rete EVs .TIMP-1 EV-derived mRNA, but further studies are needed in order to validate this hypothesis [TIMP-1 is one validated target of miR-1293, the decrease of miRNA levels observed in the EVs from the metastatic patients may be related to the upregulation of EV-derived TIMP-1 mRNA observed in the present study; however, further studies are needed in order to validate this hypothesis.Furthermore, a study performed by Cui et al. demonstrated that an increase of both endogenous and exogenous levels of TIMP-1 led to upregulation of miR-210 in a CD63-PI3K-AKT-HIF1-dependent pathway in lung adenocarcinoma cells. Moreover, upon the overexpression of TIMP-1, miR-210 was accumulated in EVs that were able to promote angiogenesis, both in vitro and in xenograft models, once in contact with recipient cells, thereby evidencing the pro-tumorigenic function of TIMP-1 . Our gropothesis . In addipothesis . Since TDespite being a pilot study involving a small group of patients, this study was able to acknowledge the potential of using EV-derived mRNAs as potential circulating biomarkers in cancer. In addition, systemic mRNA delivery, although still in its infancy, holds a huge potential for application in cancer vaccination and immunotherapy since its advantages over DNA transfection make it attractive in applications where transient expression is desired . The mRNTIMP-1 EV-derived mRNA as a prognosis biomarker for ccRCC patients with metastatic disease. Future studies involving larger cohorts of patients, with an increased follow-up period, are needed in order to further validate our hypothesis.To the best of our knowledge, this is the first study to address the potential of The present study was divided in two phases, one in vitro study and one validation study, and was conducted according to the flow chart represented in This study was conducted according to the principles of the Helsinki declaration, having been approved by the ethics committee of the Portuguese Oncology Institute of Porto . All individuals have signed written informed consent in order to participate in the study.The analysis of the EV-derived TIMP-1, TIMP-2 and MMP-1 mRNA expression was made through a hospital-based study involving a total of 61 ccRCC patients. All individuals were Caucasian from the north of Portugal, with histopathological diagnosis of ccRCC, admitted and treated at the Portuguese Oncology Institute of Porto (IPO-Porto) between November 2015 and June 2019. The patients were divided into two groups: Group A consists of 32 patients diagnosed with localized disease that were going to undergo surgical intervention; and Group B consists of patients already presenting metastatic disease. The clinical data described in Tumor classification and staging were established according to the tumor-node-metastasis (TNM) classification system of the American Joint Committee on Cancer (AJCC) 8th edition (2018) and the International Society of Urological Pathology (ISUP) Classification of Renal Neoplasia .TM, Waltham, MA, USA) with additional protocol optimizations. Briefly, cell medium was collected and centrifuged 30 min at 2000 g to remove cells and debris. After centrifugation, the supernatant was recovered and filtered through a 0.22 μm filter . Then the clarified medium and the exosome isolation reagent were mixed, in a proportion of 2:1, and incubated overnight at 4 °C. Afterwards, the mix was centrifuged 1 h at 10,000 g and the pellet containing the pre-enriched EVs was resuspended in filtered PBS and stored at −80 °C until further analysis.EVs were isolated from the cell-culture medium using the Total Exosome Isolation Reagent (from cell culture media)  with additional protocol optimizations. Firstly, 8 mL of peripheral blood was collected from the patients in EDTA tubes and centrifuged 5 min at 1800 g to obtain the plasma fraction. The plasma fraction was centrifuged three additional times at increasing speeds  for a period of 15 min each in order to obtain platelet-free plasma (PFP). After centrifugation, the supernatant was recovered and filtered through a 0.22 μm filter . A 10-min treatment with proteinase K, the Total Exosome Isolation (TEI) reagent was added to 200 μL of PFP and the solution was incubated for 30 min at 4 °C. The precipitated EVs were recovered by a 5 min centrifugation at 10,000 g at room temperature. The pellet containing the pre-enriched EVs was resuspended in filtered PBS (0.22 µm membrane filters) and stored at −80 °C until further analysis.Regarding the peripheral blood samples, EVs were isolated from the plasma fraction using the Total Exosome Isolation Kit from Plasma  system . Samples were pre-diluted in filtered PBS to achieve a concentration within the range for optimal NTA analysis. Video acquisitions were performed using a camera level of 16 and a threshold between 5 and 7. Five to nine videos of 30 s were captured per sample. Analysis of particle concentration per mL and size distribution was performed with NTA software v3.4 .9 particles of purified EVs were mixed with 40 µL of PBS containing Carboxyfluorescein Diacetate Succinimidyl Ester  in a final concentration of 40 µM and incubated for 90 min at 37 °C. For removal of unbound CFSE, Size Exclusion Chromatography (SEC) columns  were used. Samples containing unstained or stained EVs, and appropriate controls, were diluted up to 500 µL of PBS and processed by qEV following manufacturer’s instructions. EVs-enriched fractions #7, #8 and #9 were then compiled and retrieved for analysis with the Flow Cytometer Apogee A60-Micro-Plus . The A60-Micro-Plus machine is equipped with three spatially separated lasers , seven fluorescence color detectors  and three light scatter detectors . For internal control across assays, before each FC experiment, we used two mixes of beads . Before being loaded, samples were diluted in filtered PBS (0.22 µm membrane filters) to bring their concentration within the operational range of the equipment (maximum of 3000 events/s). All samples were run at a flow rate of 1.5 µL/min using a 405 nm—LALS threshold of 70. The 405 nm—LALS PMT noise level was monitored and always maintained below 0.35. For the experiments depicted, the stopping criteria utilized was the number of events acquired, so samples were run until a minimum of 250,000 events was reached. The acquired data was exported and analyzed with FlowJo software v10.4.2 .We employed EVs flow cytometry for quantification of vesicular structures in our EV isolates, as recently described by Maia et al. . In totaTM Protease & Phosphatase Inhibitor cocktail  were added, and the samples were incubated at 4 °C for 15 min. Protein was quantified using the BCA protein assay . Human MMP Antibody Array  was used to measure MMPs and TIMPs. This ELISA-like multiplex approach offers substantial benefits for examining a defined set of proteins in parallel and detects seven human MMPs  and three TIMPs . Activity was determined in EVs lysates using 100 ng of protein input. Quantification was performed by ChemiDocTM XRS+ System and data analysis was performed using Quantity One® Analysis Software .The pre-enriched EVs and cell pellets were diluted in PBS, 1X RIPA lysis buffer. Halt®, Hilden, Germany) according to the manufacturer procedure. RNA concentration and purity were measured using the NanoDrop Lite spectrophotometer  and served as a template for cDNA synthesis using a High Capacity cDNA Reverse Transcription Kit ). The thermal conditions for the cDNA synthesis were as follows: 25 °C for 10 min followed by 37 °C for 120 min and 85 °C for 5 min.RNA isolation and purification of the cells and cell-derived EVs was done using the RNeasy Plus Mini Kit , with 1X TaqManTM mRNA Expression Assays probes , and cDNA. For mRNA expression normalization two housekeeping controls were used: GAPDH  and ACTB . These housekeeping genes were chosen based on the fact that they are reported as typical EVs cargo [TM Sofware v2.2  with the same baseline and threshold set for each plate, in order to generate quantification cycle (Cq) values for all the mRNAs in each sample.mRNA expression levels were analyzed by quantitative real-time PCR. The reactions were carried out in a StepOnePlusVs cargo . The amp®, Armonk, NY, USA). According the mRNAs levels distribution, the Student t-test or Mann Whitney U test were used in order to evaluate any statistical differences in the normalized expression of the mRNAs. The quality of the housekeeping genes was tested using the BestKeeper software [TIMP-1 derived EV mRNA levels (low versus high) to the overall survival. The TIMP-1 derived EV mRNA levels low versus high levels were defined using the mean value of the –ΔCq.Statistical analyses were done using IBM SPSS Statistics software for Windows Version 22.0 (IBMsoftware . Both en


The most important factors by univariate analysis, which significantly improved the outcome of re-RT were the longer time interval between the diagnosis and second radiotherapy (p = 0.029), the lower histology grade (p = 0.034), volume of the recurrent tumour (p = 0.006) and Karnofsky performance status (KPS) (p = 0.009) at the re-irradiation. Our low fraction size re-irradiation ≥ 8 months after the first radiotherapy proved to be safe and beneficial for patients with large volume recurrent glial tumours.The aim of the present study was to evaluate the efficacy of re-irradiation (re-RT) in patients with advanced local relapses of glial tumours and to define the factors influencing the result of the hyper-fractionated external beam therapy on progression after primary management. We have analysed the data of 55 patients with brain tumours (GBM: 28) on progression, who were re-irradiated between January 2007 and December 2018. The mean volume of the recurrent tumour was 118 cm Gliomas, with incidence of 5/100 000 in adults, are the most common primary central nervous system malignancies, peaking between the fifth and sixth decades of life –4. By suIn the case of recurrence with its considerable limitations, and only if it is possible, surgical treatment has the highest efficacy . In certOn reviewing several clinical trials, the 6- and 12-month overall survival (OS-6 and OS-12), calculated from the time of re-irradiation, were 73% and 36%, respectively, whereas the 6- and 12-month of progression free survival (PFS-6 and PFS-12) were 43% and 17%, respectively –22.Between 2007 and 2018, at the Department of Oncotherapy, altogether 55 patients with recurrent glial tumours were subjected to re-irradiation. The present study has been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans. Informed consent was obtained from the patients at their first clinical admission for the anonymised use of their patient data for research purposes. According to Sect. 20/Q of No. 23/2002 Decree of the Ministry of Health, Hungary, the present study is considered as a non-interventional clinical study. The whole present study was carried out according to the ethical permission No. 4209/2018-SZTE, issued by the Ethical Committee of our University. The treatment schemes were thoroughly discussed with every single patient, independently from their actual performance status. The re-irradiation was agreed by signed informed consent. The initial care consisted of surgery in each case. The patients with grade 2 and grade 3 brain tumours received radiotherapy only postoperatively and for GBM we applied adjuvant chemoradiation therapy followed by temozolamide monotherapy up to progression. Magnetic resonance imaging (MRI) were performed three monthly. Disease progression was defined by two independent experts. At the time of diagnosis, the tumour grading was based on histological assessment. At the time of re-RT, histological evaluation was performed only in the re-operated cases, in the case of the remaining patients (without re-operation), the grading was based on clinical and radiological evaluation. The re-irradiation volume was defined on the basis of planning CT (computed tomography) and MRI fusion. Patients were immobilised with a 3-point thermoplastic mask . The planning target volume encompassed the GTV (gross tumour volume) plus 0.3-1 cm margin. The shapes of the recurrent tumours were frequently highly irregular, sometimes with multiple manifestations, and with spread to the contralateral hemisphere through the corpus callosum; or spreading along the wall of the previous surgical cavity and/or ventricle wall, resulting in larger PTV (planning target volume). The normal structures were contoured including the lens, optic chiasm, optic nerve, brain, and brainstem. Treatment planning was performed with Eclipse . The re-RT dose was 32 Gy in 1.6 Gy daily fractions in all cases, in order to avoid serious neurotoxicity. Dependent on the location and extent of the recurrent glioma, 3 DCRT or IMRT or VMAT (Rapid Arch) therapy-plans (VMAT) were generated according to the ICRU  52 recommendation. [During brain irradiation, patients received 12 mg methyl-prednisolone for prevention of brain oedema, with gradually decreased dosing after radiotherapy. The dose of methyl-prednisolone was adjusted according to the symptoms of intracranial pressure elevation due to brain oedema. The majority of the patients (32 over 23) received bevacizumab therapy after the re-RT, and these patients were controlled in a bi-weekly fashion, with physical examination up to progression and 3-months intervals MRIs were performed, whereas for the remaining group without bevacizumab treatment after re-RT, the check-ups were scheduled in 4–6 weeks. Two experts evaluated the images according to the RANO HGG (Response Assessment in Neuro-Oncology High-grade glioma) criteria . We inclTable 1 summarises the patient characteristics. The mean age of the population at the time of the primary diagnosis detection was 39 years (range: 11–71 years); 49% of them was male and 51% female. The mean age at the time of the re-irradiation was 42 years (range: 13–72 years). The KPS was in 40% of this population over 70%. At the beginning of the reirradiation, majority of the patients had minor neurological symptoms, such as hemiparesis, facial paresis, focal seizure partly controlled by antiepileptic medication, more frequently motor and sensory aphasia. Besides these symptoms, the patients preserved the ability of self-caring, except 5 patients with serious paresis needing regular help in their daily life. Out of 23 cases with repeated surgery, only four initially grade 2–3 tumours showed malignant transformation to grade 3–4. In the majority of the cases, though, reoperation took place relatively early during the course of the disease. Usually in the case of initially low grade tumours, surgery was performed prior to the first oncological management; and also grade 3 tumours were as well as re-operated some years prior to reirradiation. In 84% of the cases, based on the clinical behaviour of the tumour, their malignant transformation was highly probable at the time of reirradiation, but no regular biopsy was performed in order to confirm it. The average time interval between the diagnosis and re-irradiation was 47.4 months (range: 7.3–228 months) first and the re-irradiation was 36 months (range: 7.7–232 months) respectively. All patients received first-line systemic temozolomide treatment, either as part of initial postoperative management (GBM), or at the first relapse. 23 patients were treated with bevacizumab monotherapy, as second-line treatment. The re-irradiation was performed after the first-line systemic treatment in 45 cases, and after second-line therapy in 10 cases. The primary histological type was grade 2 astrocytoma in 15 cases, grade 3 glial tumour (anaplastic astrocytoma or oligodendroglioma) in 12 cases, and glioblastoma multiforme (GBM) in 28 cases. MGMT (O-6-methylguanine-DNA methyltransferase) methylation status was known in 26 cases. 16 patients were methylated, 9 patients borderline methylated and in one case MGMT was non-methylated.The Table 2 shows the survival data. Regarding histology, cases with lower, grade 2 malignancies had the most favourable survival values (111.0 months), whereas this value was 23 months (p < 0.001) in cases with GBM. We found a strong correlation to histological type: grade 2 astrocytoma cases had the longest survival (114.8 months), whereas the worst survival was detected of grade 4 cases .Median survival was altogether 42.6 months, as calculated from the date of the first diagnosis. The most important factors significantly influencing the outcome of re-RT were the time interval between the first and second radiotherapy, histology grade, GTV, and KPS at the re-irradiation.The mOS from the re-RT of the entire cohort was 8.4 months; 6 patients survived more than 10 months and 2 patients more than 2 years. The 6-month and the 12-month OS rate was 64% and 31% respectively.3 (range: 4.5–304 cm3). Patients with lesser than average GTV at re-RT had 12.9 months, patients with greater than average GTV at re-RT had 5.5 month of median survival (p = 0.006) , than those ones with poorer general conditions (5.6 months) Fig. .Fig. 2KaComparing time interval  with OS on univariate analysis, patients with an interval of more than 47 months from 1st to the 2nd course of RT  0.99, 95% confidence interval (CI) p = 0.029. Fig. .Fig. 3KaMedian survival, as calculated from re-irradiation for the entire group was 9.0 months. According to histopathology GBM: 6.0 months, grade 2 + 3 malignancies 10.0 months; p = 0.031.These factors proved to be significant for OS from re-RT in cox-regression univariate analysis. In multivariate analysis, the smaller GTV and better KPS remained significant influencing factors.In the present study, no significant interrelation was found between OS and age at re-RT, size of PTV, type of primary surgical intervention, or whether the second-line bevacizumab therapy preceded or followed the re-irradiation.Due to the second radiotherapy at progression, amelioration of neurological signs and KPS were experienced in 58% of our patients. Control radiological imaging detected stable disease or partial remission in 44 cases.In the majority of the cases, the prophylactic dose and escalated dose on demand of the methyl-prednisolone prevented the development of serious brain oedema and the consequent intracranial pressure elevation. We did not see any radiation necrosis on the follow up MRIs and no major cognitive deficit was associated to the re-RT assessed with MMS.In the past, re-irradiation for recurrent malignant gliomas was considered with great reluctance due to the high risk of radiation necrosis. In the recent decades after implementation of advanced RT techniques, several retrospective analyses, reviews, as well a prospective study and meta-analysis were published proving that re-irradiation is useful treatment option for recurrent brain tumours , 25, 26.3 [3 applying FSRT resulted in median survival within the range of 6.5–11 months [3 falls within the range of previously reported series [The present work represents the establishment of a careful, consequent re-RT approach with low fraction size to avoid radiation sequelae using standardised target volume and dose definition even for large volume recurrences. Selected studies on SRS for small volume recurrences of median 6.2 to 28.0 cm3 –29 showe1 months , 30–32. 1 months . However1 months . Another1 months . Analysi1 months . The sim1 months . The 9-md series , 25, 32.d series . Patientd series . It shoud series , 39. A rd series , 40. The3, radiation dose > 30–35 Gy.The evaluation of the clinical data in different series of re-RT revealed important factors, which may improve the survival, such as KPS > 70%, age < 50 years, interval > 12 months between the first RT and re-RT, target volume < 20–30 cmIn our study, significant predictors for a longer survival after re-RT were the better performance status at re-RT, the longer interval from 1st line treatment to re-RT and lower tumour grade both at diagnosis and at re-RT. The age at re-RT proved not to be a prognostic factor, however, the mean age was below 40 years. The tumour size (i.e. GTV) was one of the most significant factors for the prognosis of our patients, whilst the PTV exhibited no significant relationship to the OS. Recurrent tumour volume remained the strongest factor in multivariate analysis p = 0.038). The importance of the interval-factor is in line with former reports of re-RT. It can be assumed that the time of the first relapses after the primary treatment is an indicator of the biological behaviour of the tumour , 38, 39.. The impIn our cases, re-challenge of temosolomide was never applied, hence the primary monotherapy part was not limited in time, it was administered up to progression. Therefore, the MGMT promoter hypermethylation had less importance, because the re-irradiation was delivered when all patients developed resistance to TMZ. The MGMT status defined at the initial diagnosis was available for 27 cases, obviously with no significance on survival after re-RT. Other recent proven biological factors, such as ATRX and IDH- mutation were only partially available in our patient group.Therefore this report is limited by the lack of detailed molecular analysis as well as by the retrospective methodology which could result in a selection bias as well as an underreporting of low-grade toxicities. However, the selection bias could be reduced by the homogenous treatment concept for our cohort of patients. Nevertheless, comparison to BSC-series remains to be difficult, and conclusions about survival benefits due to intervention should be drawn with caution. Furthermore, due to the still short survival after re-irradiation, objective long-term responses after re-RT were not possible to assess for all patients.Due to the therapy, amelioration of neurological signs and KPS were experienced in 58% of our patients. Control radiological imaging detected stable disease or partial remission in 44 cases (78.6%).Although standards of salvage therapy are not yet defined for recurrent glial tumours, mainly due to paucity of high- level prospective or randomized controlled studies, re-RT of various technique is an established salvage option for selected patients .Smaller recurrent tumour size, better PS, longer interval from 1st line treatment to re-RT and lower tumour grade predict better outcome from re-RT. No radiation-associated serious adverse events were observed and the re-RT improved the performance status and neurologic symptoms in the majority of the cases. Re-irradiation with low fraction size in large volume recurrent gliomas proved to be safe and seems to be clinically beneficial in selected patient group.


Helicobacter pylori therapy.This randomized, open-label trial aimed to compare the efficacy of 10-day bismuth-containing quadruple therapy (BQT) with 7-day proton-pump inhibitor-clarithromycin containing standard triple therapy (STT) as an empirical first-line H. pylori infection were randomly assigned to either 10-day BQT  or 7-day STT . Participants who failed initial therapy were crossed over to the alternative treatment regimen. Primary outcome was the eradication rates of first-line treatment by intention-to-treat analysis.Participants with P = 0.001), modified intention-to-analysis  and per-protocol analysis . Although there was no serious adverse event, the compliance was lower with BQT than STT as a higher proportion of participants in the BQT-group discontinued therapy because of adverse events than those in the STT-group Study participants (n = 352) were randomized to receive either 10-day BQT (n = 175) or 7-day STT (n = 177). The BQT-group achieved a significantly higher eradication rate than the STT-group in the intention-to-treat analysis (74.3% vs 57.1%, respectively; H. pylori eradication in Korea.Ten-day BQT had higher eradication rates compared to that of the 7-day STT as an empirical first-line treatment for Trial registration: ClinicalTrials.gov, NCT02557932. Registered 23 September 2015, https://clinicaltrials.gov/ct2/show/NCT02557932?term=NCT02557932&draw=2&rank=1.The online version contains supplementary material available at 10.1186/s12876-021-01680-1. Helicobacter pylori, the proton pump inhibitor-clarithromycin containing standard triple therapy (STT) without antibiotic susceptibility testing is not recommended as the first-line therapy for the eradication of Helicobacter pylori in areas of high (> 15%) clarithromycin resistance [H. pylori management guidelines were first reported in 1998 [H. pylori eradication reported that STT was the most common first-line therapy prescribed (91.8%) in Korea from 2010 to 2015 [In the European and North American guidelines on treatment of f high > 5% claritsistance . In Kore in 1998 –5. A stu to 2015 . However to 2015 , 8, two  to 2015 .The revised 2013 Korean guideline recommended bismuth-containing quadruple therapy (BQT) as an alternative first-line therapy when clarithromycin resistance is suspected . The EurH. pylori infection.Thus, we conducted a randomized trial to investigate whether 10-day BQT is more effective than the 7-day STT as an empirical first-line therapy for This study was a single center, open-label, and randomized trial that was conducted at the National Cancer Center, Korea. The institutional review board at the National Cancer Center approved the trial protocol , and written informed consent was obtained from all participants before enrollment. The trial was registered with ClinicalTrials.gov (number: NCT02557932).H. pylori infection and one of following conditions: family history of gastric cancer, post-endoscopic resection state for early gastric cancer or adenoma, peptic ulcer disease, or chronic gastritis with non-ulcer dyspepsia. The exclusion criteria included the following: previous H. pylori therapy, history of gastrectomy, cancer of another organ in the last 5 years before enrollment, serious concomitant illnesses, contraindication or allergy history of trial medications, and pregnancy.Adult participants (aged ≥ 18 years) were eligible if they had confirmed H. pylori infection status of the participants at enrollment was evaluated with urea breath test, histological examination by Wright-Giemsa staining of biopsy specimens, and/or rapid urease test. Positive H. pylori infection status was confirmed as positivity of any of the following conditions: (1) urea breath test, (2) histological test of at least two biopsy sites, or (3) histological test of one biopsy site with rapid urease test.The Eligible participants were randomly assigned in a 1:1 ratio to receive either BQT for 10 days  or STT for 7 days . A web-based system using computer-generated permuted random block with a size of 2, 4, or 6 was used for randomization, and the stratification factor was sex. The randomization sequence was strictly concealed from the study investigators.H. pylori therapy was evaluated at least 6 weeks after completion of the therapy, and the urea breath test was used to determine the H. pylori status of the participants. Participants who did not achieve H. pylori eradication by the initially assigned treatment regimen, received the crossover therapy; 7-day STT for those who failed the 10-day BQT, and 10-day BQT for those who failed the 7-day STT. Another urea breath test was performed at least 6 weeks after completion of the crossover treatment.The success of H. pylori eradication rate, as assessed by the urea breath test after completion of the first-line therapy in the intention-to-treat (ITT) population. Secondary outcomes included the following: (1) H. pylori eradication rate of the first-line therapy in the modified ITT population and per-protocol (PP) population, (2) overall H. pylori eradication rate after the crossover therapy in the modified ITT population, and (3) adverse events and compliance. All adverse events were recorded according to the common terminology criteria for adverse events (CTCAE) version 4.0, and the drug compliance was evaluated by counting the returned pills of the study drugs. Serious adverse events were assessed for 30 days after the start of the assigned study therapies. The definitions of adverse event severity and serious adverse events are described in Additional file The primary study outcome was the llowing:  H. pylorH. pylori eradication rates were 74% for the 7-day STT and 86% for the 10-day BQT. The eradication rate of the 7-day STT was based on the following assumptions: (1) the clarithromycin resistance rate was 20%, (2) the eradication rates were 95% for the clarithromycin-susceptible participants and 15% for the clarithromycin-resistant participants, and (3) 7% of the participants of follow-up loss. The eradication rate for the 10-day BQT was based on the assumptions of (1) 7% loss of follow-up participants and (2) 93% eradication rate of the remaining 93% participants based on eradication rates reported by previous studies [For sample size calculation, we assumed that the  studies , 15. Witt test to analyze the continuous data. A two-sided P value of < 0.05 was considered statistically significant. All statistical analyses were performed using STATA version 16.0 .The ITT population included all participants who underwent randomization. Participants who did not perform a follow-up urea breath test were considered as eradication failures in the ITT analysis. We performed the modified ITT analysis as an ad hoc analysis, and of the participants who were included in the ITT population, those who did not start the assigned study drugs and those who did not undergo the follow-up urea breath test were excluded from the modified ITT analysis. The PP population included the participants who took at least 80% of the assigned study drugs and had a follow-up urea breath test result. Eradication rates were assessed with proportion of successful eradication and 95% confidence intervals. The safety analysis was performed for the participants who took at least one dose of the assigned treatment. We used the chi-square test or Fisher’s exact test to analyze the categorical data, and Student’s H. pylori positive participants were randomly assigned to receive either 7-day STT (n = 177) or 10-day BQT (n = 175)  (Table P < 0.001). In the PP analysis, the BQT group  had a higher eradication rate compared with the STT group .In the ITT analysis, the eradication rate with the first-line therapy was significantly higher in the participants treated with the 10-day BQT  than in those treated with the 7-day STT  Table . The posH. pylori eradication with the first-line and 33 participants who started the crossover therapy and underwent follow-up with the urea breath test after the therapy were included. In the 10-day BQT group, 130 H. pylori-eradicated participants with the first-line therapy and 13 started the crossover therapy were included. The eradication rate of the 10-day BQT as a crossover therapy was 90.9% (30/33 participants) and that of the 7-day STT was 84.6% (11/13 participants). Overall eradication rates were not significantly different between the groups  .Compliance with the study drugs was worse in participants treated with the BQT than in those treated with the STT. The proportion of the participants with low compliance, who took less than 80% of the assigned study drugs, was 26.9% (43/160 participants) in those treated with the BQT and 11.6% (19/164 participants) in participants treated with the STT (P < 0.001) .Eradication rates after the initial first-line therapy according to the study drug compliance are presented in Additional file 01) Fig. . HoweverP > 0.999)  was not different compared with those who completed ≥ 80% of that therapy  Fig. . HoweverH. pylori infection. Although the eradication rate of first-line BQT therapy was low in the ITT population (74.3%) because of the poor compliance, that in the PP population was good (92.9%). Overall combined eradication rates after the crossover therapy were similarly high in both therapy groups.This randomized trial has shown that the 10-day BQT is superior to the 7-day STT as an empirical first-line therapy for the H. pylori therapy regimens have been suggested to be ≥ 85% by the ITT analysis and ≥ 90% by the PP analysis [Our data showed that the eradication rates of the 7-day STT were 57% in the ITT analysis, 69% in the modified ITT analysis and 70% in the PP analysis. The low eradication rates are similar to those reported in a nationwide randomized trial conducted in Korea between 2016 and 2018, which reported the eradication rates of 7-day STT to be 63.9% in the ITT analysis and 71.4% in the PP analysis . Accordianalysis . ConsideIn our trial, the crossover 7-day STT in participants who failed to achieve successful eradication in the initial 10-day BQT group showed a higher eradication rate (84.6%) compared with the first-line 7-day STT (68.7%). The better eradication rate might be associated with a high compliance with the crossover 7-day STT . In addition, there might be a possibility that most of the 13 participants who received the crossover 7-day STT had clarithromycin-susceptible organism, although clarithromycin resistance was not evaluated.Our trial showed that the eradication rate of 10-day BQT was 74% in the ITT analysis, 87% in the modified ITT analysis and 93% in the PP analysis, and it was significantly higher than that of the 7-day STT as an empirical first-line therapy. The high eradication rates in our trial were consistent with those reported in previous studies investigating the first-line 10-day BQT in comparison to that of the other regimens  , 15, 18.H. pylori therapy [H. pylori infection due to chronic gastritis with non-ulcer dyspepsia. Among indications for H. pylori treatment of participants who declined to participate in the study or were lost to follow-up, the proportion of chronic gastritis with non-ulcer dyspepsia  was a higher than that of post-ESD status  and peptic ulcer disease   mod the BQT .H. pylori therapy consisting of those two regimens could provide final high eradication rates without antibiotic susceptibility testing after the crossover treatment.The resistance rates of clarithromycin or metronidazole have been reported to be high  in Korea . A randoIn our trial, treatment duration was not the same for the two therapy groups (7 days for STT and 10 days for BQT). International guidelines recommend 14 days for the STT in areas of low clarithromycin resistance , 23, andAs far as we know, this study is the first well-designed prospective randomized controlled trial comparing the 10-day BQT to the 7-day STT as the empirical first-line therapy in Korea. However, there are several limitations to this study. First, we did not evaluate the antibiotic resistance as this study was planned to evaluate the eradication rates in the empirical treatment setting. Second, the proportion of participants who did not return to follow-up visit, including withdrawal from study participation and refusal to receive the assigned study drug, was higher than expected. Thus, we also performed the ad hoc modified ITT analysis after excluding those participants.H. pylori as an empirical first-line therapy in Korea. Because the first-line 7-day STT has an unacceptable efficacy, the current Korean guidelines for H. pylori management need to be revised to include BQT as the first-line therapy.Our trial provides evidence that the 10-day BQT is highly effective in eradicating Additional file 1: Supplementary materials.


Determination of the L- and D-amino acid composition in proteins is important for monitoring process-induced racemization, and thereby protein quality loss, in food and feed. Such analysis has so far been challenging due to the need for sample hydrolysis, which generates racemization, thereby leading to an overestimation of D-amino acids. Here, validation of an LC–MS/MS-based method for the simultaneous determination of L- and D-amino acids in complex biological matrixes, like food and feed, was performed in combination with deuterated HCl hydrolysis. This approach eliminated a racemization-induced bias in the L- and D-amino acid ratios. The LC–MS/MS method was applied for the analysis of 18 free amino acids, with a quantification limit of either 12.5 or 62 ng/mL, except for D-phenylalanine, for which quantification was impaired by background interference from the derivatization agent. For hydrolyzed samples, the composition of 10 L- and D-amino acids pairs could be determined in protein. The average relative standard deviation was 5.5% and 6.1%, depending on the type of hydrolysis tubes. The method was applied on a green protein isolate (lucerne), which contained an average of 0.3% D-amino acids. In conclusion, this method allows for an unbiased analysis of L- and D-amino acid ratios in complex protein samples, such as food and feed.  Food and feed proteins usually contain a very low amount of D-amino acids. The content of D-amino acids in feed  and foodOver the last 80 years, the consumption of industrially processed foods has dramatically increased . ElevateAn increased interest in the roles of free D-amino acids in biology has resulted in the development of sensitive and high throughput analytical methods for detecting free D-amino acids. These include quantitative LC–MS methods combined with derivatization for enantiomeric separation ,16,17. HWe have developed and validated a sensitive LC–MS/MS method for the analysis of 18 free proteogenic amino acids. Combined with hydrolysis using DCl, the method allowed for simulations determination of 10 L- and D-amino acids pairs in protein. In this method, hydrogen–deuterium exchange during hydrolysis results in a +1 Da in mass increase in amino acids or peptides undergoing racemization ,20. ThisL- and D-amino acid standards were obtained from Sigma-Aldrich . Internal standards were purchased as a “cell-free” amino acid mix of 20 stable isotope-labeled amino acids . The chiral derivatization agent (S)-N-(4-nitrophenoxycarbonyl) phenylalanine methoxyethyl ester (S-NIFE) was obtained from Santa Cruz Biotechnology . All solvents for LC–MS analysis were hypergrade . Amino acid standard H , an 18 amino acid mix, was used as QC sample. Milli-Q water was used throughout the experiments . v/v) , water  and 70 mM NaOH . Mixed standard solutions were further diluted with ethanol:water  to obtain working solutions at 11 different concentration levels . Internal standard (IS) mix solution was also prepared at 1 mg/mL in ethanol:water . IS was added to calibration standards, QC and analytical samples in the ratio 1:4 (IS:sample). All solutions were kept at −80 °C until use. Stock solutions of all L- and D-amino acids were prepared individually (1 mg/mL) in the following solvents; ethanol:water  or vacuum hydrolysis tubes . For hydrolysis in glass capillary tubes, dimensions of 150 × 2.35 mm  was used. After closing one end of the glass capillary tube using a gas flame, sample was added as either a powder (protein concentrate from lucerne) or a liquid. Liquids were then evaporated to dryness using a vacuum centrifuge. Then, 40 µL of hydrolysis solution consisting of DCl  + 1% mecaptoethanol and 3% phenol  was addeFor hydrolysis in vacuum hydrolysis tubes  sample was added to the bottom of the tube and 200 µL hydrolysis liquid was added. The sample was snap-frozen in liquid nitrogen, vacuum was applied to the tube and the tube was carefully closed. v/v), respectively. All samples (both glass capillary and hydrolysis tubes) were hydrolyzed at 110 °C for 20 h. Heavy labeled internal amino acid standards (mix of 20 amino acids) were added after hydrolysis. After hydrolysis, samples were centrifuged and the supernatant was evaporated to dryness and re-dissolved in either 40 µL or 200 µL ethanol:water -NIFE in acetonitrile. The mixture was vortexed and incubated for 20 min. At this step, the reaction solution was expected to appear yellow (pH approx. 8). Undiluted hydrolyzed samples may be too acidic. In this case, more sodium tetraborate was added and the volume of Hted from .LC–MS/MS analysis was performed on a triple quadrupole tandem mass spectrometer  coupled to a 1290 Infinity LC system . Chromatographic separation was carried out on a Luna Omega C18 column   at 40 °C. Mobile phases were (A) 5% acetic acid in water and (B) 10% methanol in acetonitrile ,16. The Triple quadrupole MS conditions for analyzing S-NIFE derivatized L- and D- amino acids and the stable isotope-labeled IS were optimized in positive ion mode using multiple reaction monitoring (MRM). First, source parameter settings were optimized by injecting 3 amino acid standards: arginine, serine and phenylalanine. Optimization was achieved by injecting standards at multiple source parameter increments. The following source parameters were used: gas temperature, 350 °C; gas flow, 8 L/min; nebulizer, 15 psi; sheath gas temperature, 350 °C; sheath gas flow, 11 L/min; capillary voltage (positive mode) 3000 V; nozzle voltage (positive mode), 1500 V.Then, collision voltage (CV) and fragmentor voltage were optimized for quantifier and qualifier ions for all amino acids and there IS using direct injection and the Agilent Optimizer software, version B.08.00, built 8.0.8023.0. MRM transitions were modified from Visser et al.  using diThe method was validated in terms of linearity, lower and upper limit of quantification (LLOQ and ULOQ), stability, carry-over and with-in and between-run precision. These parameters were validated according to Guideline on bioanalytical method validation . Furthermore, the matrix effect and recovery after hydrolysis was evaluated.Calibration curves ranging from 0 to 1250 ng/mL and 1250 to 50,000 ng/mL with a total of 11 calibration points were established to cover the wide range of analysis. The linear equation and the regression coefficient was obtained from an average of 3 calibration curves . The limit of quantification (LOQ) was established by the criteria, that the individual calibration points on the standard curve should have an accuracy of ±20% relative to the linear equation.Stability of the derivatized samples was evaluated by injecting the same sample every 12 hours over a 72 h period. The samples were left in the temperature-controlled autosampler at 20 °C. Carry-over was investigated by running a MilliQ-water sample right after a calibration curve sample with the highest concentration . To establish the with-in run and between-run accuracy, 3 identical samples for each of 4 concentration levels were made. The concentration levels spanned the entire range of analysis, with 2 concentrations points on the lower range curve, approx. LLOQ and 3×LLOQ (100 ng/mL and 300 ng/mL) and with 2 concentration points on the higher range curve at approx. 30% and 75% of ULOQ . Four days later, new derivatization of the samples was made. These samples were analyzed on a new calibration curve prepared that day. n = 5). Recovery was evaluated by hydrolyzing an amino acid mix with concentrations equivalent to two calibration points  in capillary tubes without IS. After hydrolysis, IS was added and the samples were analyzed on a regular standard curve without hydrolysis. The matrix effect of hydrolyzed protein was evaluated by spike-in of 12,500 ng of standard amino acid mix into hydrolyzed α-lactalbumin  and a high range calibration curve . The linear regression coefficients, linear equations as well as LLOQ and ULOQ are listed in The LLOQ and ULOQ were defined as the lower and upper calibration points, which were less than ±20% off, relative to the linear equation . The ULOQ was equal to the highest calibration point  for all analytes, whereas the LLOQ was either 12.5 ng/mL or 62.5 ng/mL for all analytes, except phenylalanine. We observed a low baseline signal from all analytes in blank derivatized samples. In contrast, we saw a very strong background signal from phenylalanine. Consequently, we were unable to establish a linear phenylalanine calibration curve for calibration points below 12,500 ng/mL. The derivatized samples were tested for stability. The with-in run and between-run precision (%RSD) for each of the four concentrations is also listed in n = 5) and the %RSD. In general, we see a slight overestimation (4.1%–9.1%) for all amino acids. One exception is 4-hydroxy proline (2.5% underestimation), which is not present in the matrix. The %RSD was ≤5.2%. These results indicate that there is a minor matrix effect of hydrolyzed protein.In order to evaluate the matrix effect of hydrolyzed protein, we made a spike-in of 12,500 ng of each amino acid standard into a solution of a hydrolyzed pure protein. We chose α-lactalbumin, as it has an amino acid composition consisting of all the amino acids we are analyzing for, with the exception of 4-hydroxy-proline. The recovery after hydrolysis was evaluated by hydrolyzing samples from two standard point concentrations: 625 ng/mL (low-level standard curve) and 25,000 ng/mL (high-level standard curve). After hydrolysis, internal standard was added and the samples were analyzed on the regular standard curves. The results are also shown in Finally, we compared two methods of hydrolysis, using either glass capillary tubes or vacuum hydrolysis tubes. For all method validations in this paper that involved hydrolysis, the hydrolysis was performed in glass capillary tubes. As the inner diameter of these tubes is only 2.35 mm, these tubes are ideal for the hydrolysis of small volumes of liquid. However, many solid sample types, like food and feed substances, are not easily inserted into these tubes. As an alternative to glass capillary tubes, vacuum hydrolysis tubes  was used, which are ideal for the hydrolysis of larger volumes or solid samples. Therefore, we compared L- and D-amino acid analysis of a sample, hydrolyzed in either glass capillary tubes or vacuum hydrolysis tubes. For this purpose, we hydrolyzed a protein concentrate (powder) from the plant; lucerne . In The proportion of D-amino acids in lucerne was very low and did not exceed 0.62% for any amino acid. The average proportion of D-amino acids hydrolyzed in glass capillaries tubes was 0.30% D-amino acids in lucerne, relative to 0.25% D-amino acids when the hydrolysis was performed in vacuum hydrolysis tubes. In the development of our method for the simultaneous determination of the L- and D- amino acid composition in complex biological samples, we modified the LC–MS methods from previous publications on the analysis of free D-amino acids in body fluids  and tissWe observed a background signal from phenylalanine, which most likely derives from the chiral derivatization agent (S-NIFE) that holds phenylalanine in the chemical structure. From personal communication with Dr. Koning , we learWhen analyzing amino acids in intact food protein, in contrast to free amino acids, protein hydrolysis is required. Chemical hydrolysis  will result in a  racemization of the amino acids, where L-amino acids are converted to D-amino acids and vice versa . The degWe evaluated the hydrolysis method and found a minor matrix effect of hydrolyzed protein (4.1%–9.1% overestimation). In contrast, recovery after protein hydrolysis was approx. 80%–90%. Fortunately, the underestimation of the absolute quantification is partly canceled out by the matrix effect of hydrolyzed protein. More importantly, as racemization occurs evenly among L- and D-amino acids, the ratios between the enantiomers are not biased by acid hydrolysis. In the analysis of food and feed quality, it is the ratios between L- and D-amino acids, rather than the absolute values, that are important. A high D-amino acid fraction in food or feed has a negative impact on nutritional value. Acid hydrolysis is well known to be destructive to some amino acids , observeFinally, we compared two methods of hydrolysis, using either glass capillary tubes or vacuum hydrolysis tubes. Whereas glass capillary tubes are ideal for a small volume of liquid, vacuum hydrolysis tubes are needed for hydrolysis of larger volumes or solid samples. In the analysis of a protein concentrate (powder) from the plant; lucerne , we found that the proportion of D-amino acids in lucerne was very low (≤0.62% for any amino acid). The average %RSD was higher for the analysis of D-amino acids than L-amino acids  indicating that the precision of the method decreases a little at very low concentrations. Serine has the lowest difference in retention time between all the analyzed L- and D-enantiomers. The low retention time in combination with a very low proportion of D-serine in lucerne meant that the two enantiomers could not be distinguished in this analysis. However, in the analysis of other material with higher D-serine levels, the two compounds were successfully distinguished (data not shown).Industrial processing or fermentation can lead to an increase in D-amino acid levels in food and feed proteins ,2,3,4. CHere, we present a validation of an LC–MS/MS-based method for the simultaneous determination of L- and D-amino acids in complex biological matrixes, like food and feed. The use of deuterated HCl for sample hydrolysis eliminated a racemization-induced bias in the L- and D-amino acid ratios. We have adapted this method for the analysis of both small volumes of liquid and for solid samples, such as food. The method was applied to the analysis of a green protein isolate from lucerne, which is a potential alternative protein source with a low climate impact.We have developed a method for the simultaneous determination of L- and D-amino acids in proteins. Previously, LC–MS/MS methods have been developed for quantification of free D-amino acids using S-NIFE derivatization. We have adapted this approach for a precise determination of the L- and D-amino acid ratios in complex protein matrixes, such as food and feed. Importantly, hydrolysis induced biases introduced during sample preparation was eliminated by hydrogen–deuterium exchange. The method was applied to the analysis of a protein extract from lucerne, which contained an average of 0.3% D-amino acids.


The trend of MBA-CTCs over time (combining data at T0 and T1) added information with respect to separate evaluation of T0 and T1. The combined results of the two assays (MBA and CS) increased stratification accuracy, while correlation between MBA and CS was not significant, suggesting that the two assays are detecting different CTC subsets. In conclusion, this study suggests that MBA allows detection of both EpCAM-negative and EpCAM-positive, viable and label-free CTCs, which provide clinical information apparently equivalent and complementary to CS. A further validation of proposed method and cut-offs is needed in a larger, separate study.Circulating tumor cells (CTCs) belong to a heterogeneous pool of rare cells, and a unequivocal phenotypic definition of CTC is lacking. Here, we present a definition of metabolically-altered CTC (MBA-CTCs) as CD45-negative cells with an increased extracellular acidification rate, detected with a single-cell droplet microfluidic technique. We tested the prognostic value of MBA-CTCs in 31 metastatic breast cancer patients before starting a new systemic therapy (T0) and 3–4 weeks after (T1), comparing results with a parallel FDA-approved CellSearch (CS) approach. An increased level of MBA-CTCs was associated with: i) a shorter median PFS pre-therapy (123 days vs. 306;  Breast cancer is the most common cancer among women worldwide, with an estimated 2 million new cases annually . DespiteCirculating tumor cells (CTCs) are cancer cells spread from primary tumor or metastatic sites and located in body fluids. Their analysis has gained attention as one of the most promising approaches to advance clinical management of cancer, as CTCs constitute tumor material that can be accessed multiple times with minimal invasiveness, providing real-time information on the evolving biology of cancer ,4,5,6,7.The only currently FDA-approved technology for CTC enumeration in mBC is the CellSearch platform  that detects CTCs relying on the expression of the epithelial marker EpCAM on their surface. This technology has widely demonstrated the prognostic value of both the number ,13,14,15CellSearch and other similar technologies, though, are limited to the detection of CTCs expressing the epithelial marker EpCAM. However, the invasion of tumor cells from peripheral blood is a complex process, during which tumor cells can undergo epithelial-to-mesenchymal transition (EMT) that involves the downregulation of epithelial markers, such as EpCAM, cytokeratins and E-cadherin, and the upregulation of mesenchymal markers, such as vimentin and N-cadherin. Several groups have shown the existence of non-epithelial CTCs and linked these subtypes to disease progression or other clinical-pathological features ,19,20,21In this study we focused on pH dysregulation ,36,37: iDroplet microfluidics can efficiently encapsulate single cells, maintaining cell growth and proliferation properties and enabling detection of secreted molecules, as they are retained inside the droplet ,39. We pBetween September 2016 and December 2017, 31 consecutive mBC patients were recruited at the Aviano National Cancer Institute, regardless the number and type of previous lines of treatment. CTCs were enumerated using both the metabolic-based assay (MBA) and CellSearch assay (CS) before a new line of treatment (T0) and after a median of three weeks following the first therapy cycle (T1). The CONSORT diagram of evaluable patients for each method is shown in The patients’ median age was 56. Primary tumor receptor status for ER and/or PR (detected by IHC) and HER2  were positive in 21 (68%) and 3 (10%) out of 31 patients, respectively, while 8 (26%) out of 31 cases were triple-negative .Among the 31 patients recruited, 11 (35.5%) were starting their first line of therapy for metastatic disease; 28 (90.0%) received chemotherapy , 2 (6.5%) received hormone-therapy and 1 (3.2%) patient did not receive any treatment .All patients had a minimal follow-up time of 12 months (median 18 months), and the first follow-up imaging study was performed on average 5.3 ± 2 months after the T0 blood sample collection.First imaging re-evaluation after CTC enumeration documented a partial response/stable disease in 16 (59.3%) out of 27 patients with data; after the whole period of follow-up, instead, disease progression was observed in 25 (86.2%) out of 29 cases with data, and 13 (44.8%) patients deceased. Blood samples from 26 healthy donor (HD) volunteers were analyzed with MBA as negative controls.The overall strategy adopted to detect cancer cells in a background of WBCs, by measuring the extracellular acidification rate (ECAR) , is outlTo determine a pH cut-off value for detecting breast cancer cells in mBC patients, both a basal-like, triple-negative (MDA-MB-231) and a luminal, hormone receptor positive (MCF-7) breast cancer cell lines, which are considered, respectively, representative of EpCAM-low and EpCAM-high  cell linIn ex-vivo mBC blood samples, CTC count has been used to categorize patients as negative or positive for the presence of CTCs, if their count was 0 or ≥1, respectively. Moreover, a cut-off of 6 CTCs per 7.5 mL of blood was determined, adopting the same strategy that was used to set at 5 cells the prognostic cut-off value for the CS assay ,15 , with 10 patients (37.0%) being above the cut-off value of 6 cells. Overall, the average CTC count was 218 ± 1022  .Among patients scored as CTC-positive, CTCs expressing EpCAM were detected in 6 (50.0%), with an average of 84 ± 90 , whereas EpCAM-negative CTCs were detected in 10 patients (83.3%), with an average of 539 ± 1612  .p = 0.001, Mann-Whitney U-test)  out of 26, showing four and five cells, respectively . Please  U-test) .By analyzing the same mBC population by CS, we found that 17 (77.3%) out of 22 evaluable patients had at least one CTC, while eight (36.0%) patients had a CTC number above the prognostic cut-off of five cells  ,15. OverMoreover, we also determined the level of apoptotic CTCs by CS platform, as previously reported , by deteBy the MBA, 15 (57.7%) out of 26 patients presented at least one CTC, while eight (30.7%) had more than six cells. With respect to T0, the overall average number of CTC decreased from 218 ± 1022 to 37 ± 75  . EpCAM-pp = 0.2465, Wilcoxon test).Comparing CTC number at T0 and T1 for each patient, CTC level decreased in 10 cases and increased in six , while sBy CS analysis, at T1, 11 (50%) out of 22 evaluable patients had at least one detectable CTC, whereas five (23%) out 22 patients showed ≥5 CTCs. The average CTC number decreased from 129 ± 433 to 22 ± 65  .Moreover, among the CTC-positive patients, apoptotic CTCs (M30-positive) were detected in 2 (18.2%) out of 11 sample, accounting for one and seven cells, respectively. Overall, the number of M30-positive cells at both time-points was so low that it did not allow reliable data analysis.p = 0.0146, Wilcoxon test).Among the 17 patients that had CS paired samples at T0 and T1, 2 (11.8%) had an increase and 10 (58.8%) a decrease in CTC levels, while 5 (29.4%) cases showed unchanged CTC value . Unlike r = 0.39, p-value = ns; T1: Spearman r = 0.04, p-value = ns)  but not at T1   A,B; consue = ns) C,D.p-value was not significant both at T0  and T1 , indicating that the median of the two groups (MBA vs. CS) did not significantly differ.Additionally, a paired test comparing the CTC numbers detected by CS and MBA was performed . The Pratt modification to the original Wilcoxon test was used, in order to take into account the large number of cases in which both MBA and CS scored equally, detecting 0 cells. The agreement between the number of CTCs assessed at T0 and T1 and the presence of complete response/partial respose/stable disease (CR/SD/PR) or progressive disease (PD) at the first follow-up imaging study was tested. For MBA-CTCs, a good agreement (kappa = 0.761) at T0 and a poor agreement (kappa = 0.123) at T1 were observed. CS showed a moderate agreement both at T0 (kappa = 0.441) and at T1 (kappa = 0.431). In the best case (MBA-CTC at T0), 88.5% of therapy responses was correctly predicted by MBA. Results are shown in As shown in Since the MBA and the CS test are likely based on different biological peculiarities of cancer cells, the hypothesis that the two techniques might unveil additional information enabling a more accurate stratification of patients, when combined, was tested.Thus, according to the cut-off of six and five CTCs for the MBA and CS, respectively, patients were stratified into four groups: 1) Patients who had in both tests a CTC count lower than the respective reference cut-off (double < cut-off) or 2) equal or higher than it (double ≥ cut-off); 3) patient with CS-CTC ≥ cut-off, and MBA-CTC < cut-off (CS only ≥ cut-off) and 4) patients with MBA-CTCs only ≥ cut-off and CS-CTC level < cut-off (MBA only ≥ cut-off). Kaplan-Meier curves for PFS and OS are shown in p < 0.05)  B.With respect to OS, double ≥ cut-off and patients with a CTC count ≥ cut-off with only one technique had similar median OS at T0 C, but, aIt is worth noticing that, despite the two tests had a similar percentage of patients above the cut-off value, the two CTC populations were only partially overlapping: there were patients who presented a number of CTC ≥ cut-off with one test but not the other one , whereas the prevalence of patients with a CTC count ≥ cut-off in at least one test was 54.5% at T0 and 47.6% at T1. Thus, overall, the combined results of the two tests showed a ~50% increase in the prevalence of patients with CTCs ≥ cut-off with respect to that observed with each single test alone. Survival curves for these two populations (double ≥ cut-off or at least one ≥ cut-off) were almost similar but with the benefit of detecting a higher number of patients with a number of CTCs above the defined cut-off.To investigate whether a change in the level of CTCs could be used as a predictor of rapid progression and death, patients were stratified according to the CTC status before and after the start of therapy, according to the cut-off of six or five CTCs for the MBA and CS, respectively. Thus, four different groups of patients were compared: Group 1, patients having a persistently CTC count < cut-off or, Group 2, persistently ≥ cut-off; Group 3, patients with a CTC count < cut-off at T0 who converted to a CTC level ≥ cut-off post-therapy (T1); and Group 4, patients with a CTC count ≥ cut-off at T0 but who had a conversion to a CTC number < cut-off at T1 .Patients with the MBA-CTC count ≥ cut-off at both time-points had the worst prognosis compared to patients with a CTC count < cut-off at both time-points. Patients with CTC count ≥ cut-off either at baseline or at follow-up had an intermediate and statistically different outcome compared to Group 1 and Group 2 A. In CS As shown in Studies of other groups involving CTC detected with a metabolism-based approach did not correlate results with clinical outcome. In the present pilot study, we determined that elevated MBA-CTCs are linked to shorter overall and progression-free survival. Additional information was obtained considering CTC levels over time.One of the limitations of this study is the relatively small cohort involved, though our cohort reflects general population in terms of cancer subtype prevalence , metastatic sites and lines of therapy. This is further confirmed by the results obtained in this study with CS, which are aligned with larger clinical trials in metastatic breast, colon and prostate cancers ,16,42,43Another limitation is the relatively small volume of sample analyzed . We calculated and analyzed cumulative Poisson distribution for known CTC concentrations ranging from 2 to 30 cells/7.5 mL to evaluate how a smaller sampling volume could affect accuracy of CTC estimation. The analysis is descripted in Interestingly, the agreement between cells detected by MBA and CS was relatively weak and no significant correlation was found, while both methods independently yielded similar prognostic significance. Moderate correlation and fair agreement were found instead for EpCAM-positive cells detected by the two methods suggesting partial overlapping of EpCAM population. In other words, it seems like some, but not all, of EpCAM-positive CK-positive cells display anormal metabolism, while some, but not all, of EpCAM-positive MBA-positive cells display cytokeratins. By combining data obtained by MBA and CS, additional information could be gained, suggesting that the two techniques detect different CTC subgroups with complementary information, possibly reflecting different aspects of disease evolution. This interpretation is strengthened by the fact that the prevalence of single-positive patients (either MBA or CS above cut-off) is roughly 50% higher than the prevalence obtained considering single MBA or CS testing. A possible explanation is the fact that metabolism is altered also in non-epithelial cells , which aIt should be now clear that referring to CTCs without further definition might be confusing, as it is a rather blurred group of cells of different nature and maybe distinct clinical implications. More studies are needed to clearly understand if different types of cells could be linked to specific clinical and pathological features or therapy susceptibilities.Hypothesizing a predictive role of MBA-CTCs, a growing body of evidence suggests that the acidic extracellular environment of tumor tissues confers chemoresistance: indeed, a majority of chemotherapeutics  behaves like weak bases which are quickly protonated in low pH environments, leading to a reduced cellular uptake and treatment efficacy . Thus, rCTCs identified by their aberrant metabolism seem at least as tightly linked to survival and disease progression as epithelial CTCs, either when measured before or after the therapy, and even more when considered over time; it emerged that MBA-CTCs provide different information with respect to CS-CTC, which can be combined to increase accuracy in mBC prognostic stratification. Overall, this pilot study can be used as a foundation to design larger clinical studies needed to confirm and validate MBA-CTCs as prognostic/predictive biomarkers.This pilot study trial was conducted comparing the metabolism-based assay (MBA) with the CellSearch (CS) system to test prognostic meaning of CTC enumeration. The study was conducted at the IRCCS-CRO Aviano-National Cancer Institute and approved by the Institutional Review Board with number IRB-12-2014. Informed and written consent was obtained from all patients and healthy donors before their enrolment, and their clinico-pathological information was recorded. Thirty-one patients with progressive and measurable mBC, at the beginning of a new systemic therapy, without limits to round and type of previous therapies  were included. All patients had an Eastern Cooperative Oncology Group performance status (ECOG PS) score ≤ 1.Before starting a new therapy, patients underwent a baseline (T0) blood drawn for CTC evaluation and routine clinical tests. Another blood sample was collected 3–4 weeks after the beginning of the therapy . Among the 31 enrolled patients, 27 and 26 blood samples were evaluated for the presence of CTC with the MBA at T0 and T1, respectively, whereas CTC count was evaluable with the gold standard CS for 22 patients at both time points. Details about reasons for exclusion  are summarized in Clinical re-evaluation of the disease status was conducted depending on the type and schedule of the therapy; Standard Response Evaluation Criteria in Solid Tumors (RECIST) criteria were used to determine patients’ responses to treatment. This study did not interfere with routine imaging schedule, nor imposed a uniform imaging schedule to all patients. The follow-up imaging schedule was decided by the oncologists on an individual basis. For most patients entered in the study, CT of the chest and abdomen, or PET, approximately every 3–6 months, were performed. In selected cases e.g., in the case of liver metastases as the unique site of disease, the disease parameter was simplified by using a liver ultrasound.In this work, a microfluidic device was employed for droplet generation, and a second one for droplet reinjection in order to screen their fluorescence for pH determination and antibody staining. Each device was made of PDMS bonded to a glass surface, as previously reported .The optical setup for measuring droplet fluorescence consisted of an IX70 inverted fluorescence microscope . A 25 mW, 405 nm laser beam was focused with a cylindrical lens crossing orthogonally the microfluidic channel. Fluorescence signal emitted from droplets was captured by a 40× objective , split with dichroic filter and detected through bandpass filters  by photomultiplier tubes (PMTs) . Signal went through a transimpedance amplifier with 1V/uA gain and detected by the acquisition system  with a 10 μsec scan rate.The emulsification device was used to generate droplets. This device has a flow-focusing junction, where the aqueous phase (cell suspension) and carrier oil stream meet, and droplet generation occurs (forming a water-in-oil emulsion). The resulting emulsion flowed off-chip through PTFE tubing  connected to the outlet of the device and collected into 1.5 mL vial  placed on an ice-cold rack . The vial containing droplets was incubated for the desired incubation time at 37 °C. After incubation, droplets were immediately cooled by replacing the vial on the ice-cold rack and reinjected into the required microfluidic device for subsequent analysis.Droplets were reinjected into the emulsion device through PTFE tubing . The data acquisition system has a 10 μsec scan rate. The signal output voltage of each droplet was recorded and processed in real time by a field programmable gate array (FPGA) card. Droplets with a size within defined range and gated fluorescence were imaged by synchronous triggering of an LED and a CMOS camera, and  sorted with a dielectrophoretic pulse. Data are transferred from the FPGA to the real-time controller providing a record of images and raw tracks for each run. A custom LabVIEW software allows the operator to access the picture gallery, evaluate the pictures of droplets to discard droplets that are empty or containing particles not compatible with the morphology of intact cells (debris).The pH-sensitive fluorescent dye SNARF-5F  was used to measure the pH of each droplet. SNARF-5F responds to pH variations by undergoing a wavelength shift in the emission spectra. Such pH-dependent shifts allow the ratio of the fluorescence intensities from the dye at two emission wavelengths (580 nm and 630 nm) to be used for quantitative determination of pH. For each droplet the ratio of emitted fluorescence intensities at 580 and 630 nm (580/630 ratio) of SNARF-5F is calculated in real time. As the pH is more acidic, SNARF-5F fluorescence increases at 580 nm while decreases at 630 nm. To calibrate the system, Joklik’s EMEM medium titrated at different pH  was added with 4 µM SNARF-5F, emulsified and droplets screened for fluorescence. The 580/630 nm ratio were calculated for each pH and plotted against the respective pH, which allowed constructing a calibration curve.2. Cells used were detached before confluency, tested for absence of mycoplasma contamination and validated for short tandem repeat profiling.Breast cancer cell lines MDA-MB-231 and MCF7 were obtained from the American Type Culture Collection  and cultured in DMEM medium  supplemented with 10% FBS, in a humidified atmosphere at 37 °C and 5% COTo identify a threshold of extracellular acidification rate  able to better discriminate between CTCs and WBCs, the ECAR of breast cancer cell lines (MDA-MB-231 and MCF7), and WBCs was separately assessed by using the MBA. WBCs were obtained from the peripheral blood of healthy donors (n = 3), after red blood cell lysis with BD Pharm Lyse lysing solution  according to the manufacturer’s instructions, while breast cancer cell line (MCF7 and MDA-MB-231), grown as previously described at a confluence of 70–80%, were detached with 1% Trypsin-EDTA. Cells were resuspended at a concentration of 1,000,000 cells/mL in 50 μL of the unbuffered Joklik’s modified EMEM culture medium (Sigma) containing 2 mM EDTA, 0.1% BSA, 15% Optiprep and 4 mM of the fluorescent pH indicator SNARF-5F, emulsified as described in the previous section, incubated at 37 °C for 30 min and analyzed. All experiments were performed in triplicate at various cell passages.2 for 30 min and then reinjected into the microfluidic channel for fluorescent reading of pH, CD45 and EpCAM expression, and the acquisition of bright-field imaging of each positive event. Positive events were defined as CD45-negative cells able to acidify their extracellular environment to a pH lower than 6.4 (MBA-CTC). The number of CTCs was then proportionally adjusted to 7.5 mL for an easier comparison to the one reported by the CS test. This was done by obtaining the total volume of sample analyzed  or adverse events (clogging of microfluidics and restart of data acquisition). Fortunately, droplet microfluidics technology allows the operator to know the precise volume analyzed, because the number of total droplets analyzed and the volume of droplet is known. Therefore, the operator can always calculate the proportion of detected events with respect to the analyzed volume)  and doing the proper proportion (MBA-CTC (measured)/volume analyzed = MBA-CTC/7.5 mL). All evaluations were performed without knowledge of the clinical status of the patients.Blood samples were drawn into K2-EDTA Vacutainer tubes (Beckton Dickinson) and maintained at room temperature. For each sample, 2.5 mL of blood were analyzed within 2 h after collection. Red blood cells (RBC) were lysed with the BD Pharm Lyse lysing solution , according to the manufacturer’s instructions, and centrifuged at 200 RCF for 5 min. Thereafter, the nucleated fraction was depleted of CD45-positive WBCs and residual RBCs using CD45 and Glycophorin A microbeads , respectively, and LD separation columns in a MACS MIDI separator (Miltenyi Biotec), according to the manufacturer’s instructions. The CD45-negative cells were recovered, centrifuged at 300 RCF for 10 min and stained with anti-CD45  and anti-EpCAM  for 25 min at room temperature. After washing the sample with PBS-BSA 0.5%, cells were resuspended in 50 μL of the unbuffered Joklik’s modified EMEM culture medium (Sigma) containing 2 mM EDTA, 0.1% BSA, 15% Optiprep and 4mM of the fluorescent pH indicator SNARF-5F (Thermo Fisher Scientific). Then, cells were single-cell encapsulated in monodispersed droplets using the droplet microfluidic platform as described in previous section. The emulsion was collected and incubated at 37 °C and 5% COBlood samples were drawn into 10 mL CellSave Preservative Tubes (Menarini) and maintained at room temperature. Blood samples were sent to Veneto Institute of Oncology IOV-IRCCS, processed and analyzed according to manufacturer’s instruction . To quanp < 0.05 was considered significant.Data were analyzed using GraphPad Prism 6 . Patient and clinical characteristics were presented as frequency and percentage, median and range, mean and standard deviation (SD), as appropriate. Comparison of median between groups were performed by Mann-Whitney test and groups were compared using the Wilcoxon rank test, as appropriate. PFS and OS  were determined by Kaplan–Meier plots, with data being censored at last follow-up if progression or death had not occurred. Gehan-Breslow-Wilcoxon test were used to compare the survival curves by CTC detection groups. Median PFS times and Cox proportional hazard ratios over all possible CTC cut-offs using baseline data were used to determine the optimal CTC cut-off for the prediction of PFS. The median PFS for the patients above or below each cut-off was calculated. In addition, the percentage of patients above each CTC cut-off was calculated. The best cut-off was selected as the one with the highest Cox hazard ratio, being above the normal background and having at least 40% patients above the cut-off. A CTC count of six or more per 7.5 mL of blood is predictive of shorter PFS. The chart showing median PFS for each cut-off is shown in In this pilot study, cellular pH dysregulation is exploited in an attempt to detect CTCs in metastatic breast cancer. Results suggest that metabolically aberrant CTCs, detected with the method presented herein, provide useful prognostic information. Clinical validity needs further confirmation in larger cohorts of patients.Patent number ITRM20130700A1, 19 Dec 2013. Patent family ID 50073355 .